Abstract Immunotherapy approaches for cancer can result in occasional long term remissions of advanced metastatic cancers resistant to conventional modes of therapy. Development of more effective approaches requires a detailed analysis of the immunobiology of these antitumor responses. However, existing technologies for the study of immune responses have proven to have limited ability to fully characterize this complex process, manily due to the fact that the cells that orchestrate these responses, the tumor antigen-specific CD8+ cytotoxic T lymphocytes (CTL) are rare cells in peripheral circulation. We propose to develop a modular technology to allow for the design, production and validation of functional components for the enumeration of tumor-specific CD8+ CTL using limiting samples, eventually allowing to quantitate and functionally characterize tumor-infiltrating CD8+ CTL. In Aim 1 we propose to use capture MHC tetramers loaded with tumor antigens (the specific ligands for CD8+ CTL) to develop murine and human micro-lmmunochip (uIC) modules with digitally controlled, individually accessible microfluidic chambers to capture, sort, quantitate and incubate tumor-specific T-cells. In Aim 2 we propose to develop individual modules for quantifying surface, intracellular and secreted proteins, from very small numbers of anti-tumor specific T cells (even single cells), and a module for quantifying mRNA signatures from small numbers of cells (in collaboration with Fluidigm). In Aim 3 the different modules will be integrated and tested, first using defined populations of tumor-specific CD8+ CTL, followed by samples of increasing complexity obtained from mouse models of immunotherapy, and eventually human tumor-specific CD8+ CTL obtained from patients participating in clinical trials of cancer immunotherapy being conducted at UCLA.